Fluorescence-based reporters for mutagenesis detection in e. coli

ABSTRACT

Direct detection of mutagenesis in prokaryotes by reversion of an inactivating mutation (reversion mutation assay), producing a quantitative signal for in vivo mutagenesis, may greatly reduce the amount of test chemicals and labor involved in these assays. Further, transcriptional coupling of β-lactamase reversion and GFP, translational fusion between β-lactamase and GFP with stop codon in GFP, and a novel dual reporter to monitor continuous mutagenesis may be used in methods described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/473,442, filed on Jun. 25, 2019, which is the National Phaseunder 35 U.S.C. § 371 of PCT International Application No.PCT/US2017/068410 which has an international filing date of Dec. 26,2017, and which claims priority to U.S. Provisional Application No.62/441,411, filed on Jan. 1, 2017, the entire contents of each of whichare hereby incorporated by reference.

STATEMENT OF SUPPORT

This disclosure was made with government support under the followinggrants: National Institute of Environmental Health Sciences) grantnumber RO1ES019625. The government has certain rights in the disclosure.

FIELD OF THE INVENTION

The disclosure relates to the direct detection of mutagenesis inprokaryotes using detection of reporter inactivation (forward mutationassay) and reversion of an inactivating mutation (reversion mutationassay).

BACKGROUND OF THE INVENTION

Mutagenesis following exposure to chemicals can be used to detectgenotoxicity, which is an indicator of the potential of the chemical tocause cancer and/or birth defects. Mutagenesis assays are also used as areadout to study processes of DNA replication, DNA repair, DNA damagetolerization, and mechanisms of DNA homeostasis.

BRIEF SUMMARY OF THE INVENTION

Mutagenesis in model organisms following exposure to chemicals can beused as an indicator of genotoxicity. Mutagenesis assays can also beused to study mechanisms of DNA homeostasis. In prokaryotes, there aretwo approaches to the detection of mutagenesis: reporter inactivation(which is the basis for forward mutation assays) and reversion of aninactivating mutation (which occurs in reversion mutation assays). Bothmethods are labor-intensive and require visual screening, thequantification of colonies on solid media, or the determination of aPoisson distribution in liquid culture. Disclosed herein are reversionreporters that can be used to measure mutagenesis in vivo. Thesereporters produce a quantitative output. As a result, a mutagenesisassay using these reporters can be performed with a smaller amount ofreagent, in less time, and with less labor. The assay involves a βlactamase (TEM-1)-based reversion assay and a fluorescent protein suchas GFP or a derivative thereof

Nucleic acids encoding the TEM-1 and the fluorescent protein can beprovided on the same plasmid. Alternatively, they can be fused duringprotein translation with the N-terminus of the ORF interrupted using astop codon. Also disclosed herein is a reporter that monitors continuousmutagenesis in mutator strains of bacteria. This reporter involves tworeversion markers and has the benefit of allowing the detection of thetwo mutation events in real time. Disclosed are the reporter systems,methods of using the reporters, and a demonstration of key features ofthe reporters.

In one aspect, the disclosure features a method of detecting mutagenesisin E. coli, the method comprising: (a) culturing E. coli cells in afirst liquid culture at a restrictive temperature, wherein the E. colicells in the first liquid culture comprise a plasmid comprising (i) afirst polynucleotide encoding an inactive β-lactamase and having atleast 90% sequence identity to a sequence of any one of SEQ ID NOS: 2-7,wherein nucleotides 202 to 204 of the sequence of any one of SEQ ID NOS:2-7 does not encode serine, and (ii) a second polynucleotide encoding afluorescent protein, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter; (b) plating the E.coli cells in the first liquid culture on a solid media comprising anantibiotic; (c) incubating the first solid media at a permissivetemperature that allow the growth of E. coli colonies; (d) selecting afluorescent E. coli colony from the solid media; (e) culturing thefluorescent E. coli colony in a second liquid culture at a permissivetemperature, wherein the second liquid culture comprises the antibiotic;and (f) measuring the change in growth of the E. coli cells of thesecond culture relative to the first liquid culture, wherein the changein growth indicates mutagenesis of the inactive β-lactamase to an activeβ-lactamase.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In another aspect, the disclosure features a method of detectingmutagenesis in E. coli, the method comprising: (a) culturing E. colicells in a first liquid culture at a restrictive temperature, whereinthe E. coli cells in the first liquid culture comprise a plasmid,wherein the plasmid comprises (i) a first polynucleotide encoding anon-fluorescent protein, and (ii) a second polynucleotide encoding anactive β-lactamase, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter; (b) plating the E.coli cells in the first liquid culture on a solid media comprising anantibiotic; (c) incubating the first solid media at a permissivetemperature that allow the growth of E. coli colonies; (d) selecting afluorescent E. coli colony from the solid media; (e) culturing thefluorescent E. coli colony in a second liquid culture at a permissivetemperature, wherein the second liquid culture comprises the antibiotic;and (f) measuring the change in fluorescence of the second liquidculture relative to the first liquid culture, wherein the change influorescence indicates mutagenesis of the non-fluorescent protein to afluorescent protein.

In some embodiments of this aspect, the active β-lactamase has asequence of SEQ ID NO: 8.

In another aspect, the disclosure features a method of detectingmutagenesis in E. coli, the method comprising: (a) culturing E. colicells in a first liquid culture at a restrictive temperature, whereinthe E. coli cells in the first liquid culture comprise a plasmid,wherein the plasmid comprises (i) a first polynucleotide encoding aninactive β-lactamase and having at least 90% sequence identity to asequence of any one of SEQ ID NOS: 2-7, wherein nucleotides 202 to 204of the sequence of any one of SEQ ID NOS: 2-7 does not encode serine,and (ii) a second polynucleotide encoding a non-fluorescent protein,wherein the first polynucleotide and the second polynucleotide areoperably linked to a promoter; (b) plating the E. coli cells in thefirst liquid culture on a first solid media comprising an antibiotic;(c) incubating the first solid media at a permissive temperature thatallow the growth of E. coli colonies; (d) selecting a non-fluorescent E.coli colony from the first solid media; (e) culturing thenon-fluorescent E. coli colony in a second liquid culture at arestrictive temperature, wherein the second liquid culture comprises theantibiotic; (f) plating the E. coli cells in the second liquid cultureon a second solid media comprising the antibiotic; (g) incubating thesecond solid media at a permissive temperature that allow the growth ofE. coli colonies; (h) selecting a fluorescent E. coli colony from thesecond solid media; (i) culturing the fluorescent E. coli colony in athird liquid culture at a permissive temperature, wherein the thirdliquid culture comprises the antibiotic; and (j) measuring the change influorescence of the third liquid culture relative to the first liquidculture, wherein the change in fluorescence indicates mutagenesis of theinactive β-lactamase to an active β-lactamase and of the non-fluorescentprotein to a fluorescent protein.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In some embodiments of any of the aspects above, the firstpolynucleotide is located 5′ to the second polynucleotide in theplasmid. In some embodiments, the plasmid further comprises a linkerbetween the first polynucleotide and the second polynucleotide. Inparticular embodiments, the linker may have a sequence of SEQ ID NO: 44.

In some embodiments, the antibiotic is a β-lactam antibiotic selectedfrom the group consisting of kanamycin, carbenicillin, benzathine,benzylpenicillin, penicillin G, penicillin V, procaine,benzylpenicillin, cloxacillin, dicloxacillin, flucloxacillin,methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin,mecillinam, carboxypenicillins, ticarcillin, ureidopenicillins,azlocillin, mezlocillin, piperacillin, cephalosporin C, cefoxitin,cephalosporin, cephamycin, cefazolin, cephalexin, cephalosporin C,cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin,cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime,cefpirome, ceftaroline, thienamycin, biapenem, doripenem, ertapenem,faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, andthienamycin. In particular embodiments, the antibiotic is kanamycin orcarbenicillin.

In some embodiments of the aspects described herein, the β-lactamase isTEM-1.

In some embodiments, the fluorescent protein comprises GFP or aderivative thereof. In particular embodiments, the fluorescent proteincomprises a sequence of any one of SEQ ID NOS: 9 and 10.

In some embodiments, the non-fluorescent protein comprises a sequence ofSEQ ID NO: 11. In some embodiments, the non-fluorescent protein may becreated by mutating codon ACT which encodes threonine at position 49 ofSEQ ID NO: 9 to codon GCT which encodes alanine. In some embodiments,the non-fluorescent protein may be created by mutating codon TGC whichencodes cysteine at position 48 of SEQ ID NO: 9 to codon TAC whichencodes tyrosine. In some embodiments, the non-fluorescent protein maybe created by mutating codon CCA which encodes proline at position 56 ofSEQ ID NO: 9 to codon CTA which encodes leucine. In some embodiments,the non-fluorescent protein may be created by mutating codon GTC whichencodes valine at position 61 of SEQ ID NO: 9 to codon GAC which encodesaspartic acid. In some embodiments, the non-fluorescent protein may becreated by mutating codon ACT which encodes threonine at position 62 ofSEQ ID NO: 9 to codon GCT which encodes alanine. In some embodiments,the non-fluorescent protein may be created by mutating codon CCC whichencodes proline at position 89 of SEQ ID NO: 9 to codon TCC whichencodes serine. In some embodiments, the non-fluorescent protein may becreated by mutating codon GGT which encodes glycine at position 91 ofSEQ ID NO: 9 to codon GAT which encodes aspartic acid. In someembodiments, the non-fluorescent protein may be created by mutatingcodon TAT which encodes tyrosine at position 92 of SEQ ID NO: 9 to codonTGT which encodes cysteine. In some embodiments, the non-fluorescentprotein may be created by mutating codon GAC which encodes aspartic acidat position 103 of SEQ ID NO: 9 to codon GTC which encodes valine. Insome embodiments, the non-fluorescent protein may be created by mutatingcodon TAC which encodes tyrosine at position 143 of SEQ ID NO: 9 tocodon TGC which encodes cysteine. In some embodiments, thenon-fluorescent protein may be created by mutating codon CAA whichencodes glutamine at position 183 of SEQ ID NO: 9 to codon CAC whichencodes histidine. In some embodiments, the non-fluorescent protein maybe created by mutating codon GAA which encodes glutamic acid at position213 of SEQ ID NO: 9 to codon GGA which encodes glycine.

In some embodiments of the aspects of the disclosure, the firstpolynucleotide and the second polynucleotide are expressed as a fusionprotein. In some embodiments, the fusion protein comprises a sequence ofSEQ ID NO: 12.

In some embodiments, the methods further comprise exposing the E. colicells to a test compound added to the first liquid culture and/or secondliquid culture. In some embodiments, the test compound is a mutagen.

In another aspect, the disclosure features a kit comprising a plasmidcomprising (i) a first polynucleotide encoding an inactive β-lactamaseand having at least 90% sequence identity to a sequence of any one ofSEQ ID NOS: 2-7, wherein nucleotides 202 to 204 of the sequence of anyone of SEQ ID NOS: 2-7 does not encode serine, and (ii) a secondpolynucleotide encoding a fluorescent protein, wherein the firstpolynucleotide and the second polynucleotide are operably linked to apromoter.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In another aspect, the disclosure features a kit comprising a plasmidcomprising (i) a first polynucleotide encoding a non-fluorescentprotein, and (ii) a second polynucleotide encoding an activeβ-lactamase, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter.

In another aspect, the disclosure features a kit comprising a plasmidcomprising (i) a first polynucleotide encoding an inactive β-lactamaseand having at least 90% sequence identity to a sequence of any one ofSEQ ID NOS: 2-7, wherein nucleotides 202 to 204 of the sequence of anyone of SEQ ID NOS: 2-7 does not encode serine, and (ii) a secondpolynucleotide encoding a non-fluorescent protein, wherein the firstpolynucleotide and the second polynucleotide are operably linked to apromoter.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In another aspect, the disclosure features a kit comprising E coli cellstransformed with a plasmid comprising (i) a first polynucleotideencoding an inactive β-lactamase and having at least 90% sequenceidentity to a sequence of any one of SEQ ID NOS: 2-7, whereinnucleotides 202 to 204 of the sequence of any one of SEQ ID NOS: 2-7does not encode serine, and (ii) a second polynucleotide encoding afluorescent protein, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In another aspect, the disclosure features a kit comprising E coli cellstransformed with a plasmid comprising (i) a first polynucleotideencoding a non-fluorescent protein, and (ii) a second polynucleotideencoding an active β-lactamase, wherein the first polynucleotide and thesecond polynucleotide are operably linked to a promoter.

In another aspect, the disclosure features a kit comprising E coli cellstransformed with a plasmid comprising (i) a first polynucleotideencoding an inactive β-lactamase and having at least 90% sequenceidentity to a sequence of any one of SEQ ID NOS: 2-7, whereinnucleotides 202 to 204 of the sequence of any one of SEQ ID NOS: 2-7does not encode serine, and (ii) a second polynucleotide encoding anon-fluorescent protein, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter.

In some embodiments of this aspect, nucleotides 202 to 204 of thesequence of any one of SEQ ID NOS: 2-7 encodes proline, threonine,arginine, a stop codon, or asparagine.

In some embodiments, the E. coli cells in the kit are of a mutatorstrain and/or a readout strain.

In some embodiments, the first polynucleotide is located 5′ to thesecond polynucleotide in the plasmid.

In some embodiments, the plasmid further comprises a linker between thefirst polynucleotide and the second polynucleotide. In particularembodiments, the link has a sequence of SEQ ID NO: 44.

In some embodiments, the β-lactamase is TEM-1.

In some embodiments of the aspects directed to kits of the disclosure,the fluorescent protein comprises GFP or a derivative thereof. Inparticular embodiments, the fluorescent protein comprises a sequence ofSEQ ID NOS: 9 and 10.

In some embodiments of the aspects directed to kits of the disclosure,the non-fluorescent protein comprises a sequence of SEQ ID NO: 11. Insome embodiments, the non-fluorescent protein may be created by mutatingcodon ACT which encodes threonine at position 49 of SEQ ID NO: 9 tocodon GCT which encodes alanine. In some embodiments, thenon-fluorescent protein may be created by mutating codon TGC whichencodes cysteine at position 48 of SEQ ID NO: 9 to codon TAC whichencodes tyrosine. In some embodiments, the non-fluorescent protein maybe created by mutating codon CCA which encodes proline at position 56 ofSEQ ID NO: 9 to codon CTA which encodes leucine. In some embodiments,the non-fluorescent protein may be created by mutating codon GTC whichencodes valine at position 61 of SEQ ID NO: 9 to codon GAC which encodesaspartic acid. In some embodiments, the non-fluorescent protein may becreated by mutating codon ACT which encodes threonine at position 62 ofSEQ ID NO: 9 to codon GCT which encodes alanine. In some embodiments,the non-fluorescent protein may be created by mutating codon CCC whichencodes proline at position 89 of SEQ ID NO: 9 to codon TCC whichencodes serine. In some embodiments, the non-fluorescent protein may becreated by mutating codon GGT which encodes glycine at position 91 ofSEQ ID NO: 9 to codon GAT which encodes aspartic acid. In someembodiments, the non-fluorescent protein may be created by mutatingcodon TAT which encodes tyrosine at position 92 of SEQ ID NO: 9 to codonTGT which encodes cysteine. In some embodiments, the non-fluorescentprotein may be created by mutating codon GAC which encodes aspartic acidat position 103 of SEQ ID NO: 9 to codon GTC which encodes valine. Insome embodiments, the non-fluorescent protein may be created by mutatingcodon TAC which encodes tyrosine at position 143 of SEQ ID NO: 9 tocodon TGC which encodes cysteine. In some embodiments, thenon-fluorescent protein may be created by mutating codon CAA whichencodes glutamine at position 183 of SEQ ID NO: 9 to codon CAC whichencodes histidine. In some embodiments, the non-fluorescent protein maybe created by mutating codon GAA which encodes glutamic acid at position213 of SEQ ID NO: 9 to codon GGA which encodes glycine.

In some embodiments of the aspects directed to kits of the disclosure,the first polynucleotide and the second polynucleotide are expressed asa fusion protein. In particular embodiments, the fusion proteincomprises a sequence of SEQ ID NO: 12.

Definitions

As used herein, the term “polynucleotide” refers to an oligonucleotide,or nucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand.

As used herein, the term “promoter” refers to a polynucleotide sequencecapable of driving transcription of a coding sequence in a cell. Thus,promoters may include cis-acting transcriptional control elements andregulatory sequences that are involved in regulating or modulating thetiming and/or rate of transcription of a gene. For example, a promotercan be a cis-acting transcriptional control element, including anenhancer, a promoter, a transcription terminator, an origin ofreplication, a chromosomal integration sequence, 5′ and 3′ untranslatedregions, or an intronic sequence, which are involved in transcriptionalregulation. These cis-acting sequences typically interact with proteinsor other biomolecules to carry out (turn on/off, regulate, modulate,etc.) gene transcription.

As used herein, the term “plasmid” refers to an extrachromosomalcircular DNA capable of autonomous replication in a given cell. Incertain embodiments, the plasmid is designed for amplification andexpression in bacteria (e.g., E. coli). Plasmids can be engineered bystandard molecular biology techniques. See Sambrook et al. LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989),N.Y. A plasmid may be a recombinant DNA molecule containing a desiredcoding sequence and appropriate nucleic acid sequences necessary forexpression of the operably linked coding sequence in a particular hostcell. Nucleic acid sequences necessary for expression in prokaryotesusually include a promoter, an operator (optional), and a ribosomebinding site, often along with other sequences.

As used herein, the term “operably linked” refers to nucleic acidsequences or proteins that are placed into a functional relationshipwith another nucleic acid sequence or protein. For example, a promotersequence is operably linked to a coding sequence (e.g., the firstpolynucleotide and the second polynucleotide in a plasmid) if thepromoter promotes transcription of the coding sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, although they need not be, and that a gene and a regulatorysequence (e.g., a promoter) are connected in such a way as to permitgene expression.

As used herein, the term “percent (%) sequence identity” refers to thepercentage of nucleic acid or amino acid residues of a candidatesequence that are identical to the nucleic acid or amino acid residuesof a reference sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent identity (i.e., gapscan be introduced in one or both of the candidate and referencesequences for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). Alignment for purposes ofdetermining percent identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. In someembodiments, the percent nucleic acid or amino acid sequence identity ofa given candidate sequence to, with, or against a given referencesequence (which can alternatively be phrased as a given candidatesequence that has or includes a certain percent nucleic acid or aminoacid sequence identity to, with, or against a given reference sequence)is calculated as follows:

100×(fraction of A/B)

where A is the number of nucleic acid or amino acid residues scored asidentical in the alignment of the candidate sequence and the referencesequence, and where B is the total number of nucleic acid or amino acidresidues in the reference sequence. In some embodiments where the lengthof the candidate sequence does not equal to the length of the referencesequence, the percent nucleic acid or amino acid sequence identity ofthe candidate sequence to the reference sequence would not equal to thepercent amino acid (or nucleic acid) sequence identity of the referencesequence to the candidate sequence.

In particular embodiments, a reference sequence aligned for comparisonwith a candidate sequence may show that the candidate sequence exhibitsfrom 50% to 100% identity across the full length of the candidatesequence or a selected portion of contiguous nucleic acid or amino acidresidues of the candidate sequence. The length of the candidate sequencealigned for comparison purpose is at least 30%, e.g., at least 40%,e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of thereference sequence. When a position in the candidate sequence isoccupied by the same nucleic acid or amino acid residue as thecorresponding position in the reference sequence, then the molecules areidentical at that position.

For example, as described herein, a plasmid in methods of the disclosuremay comprise a polynucleotide encoding an inactive β-lactamase andhaving at least 90% sequence identity to a sequence of any one of SEQ IDNOS: 2-7, wherein nucleotides 202 to 204 of the sequence of any one ofSEQ ID NOS: 2-7 does not encode serine. In some embodiments, thepolynucleotide encoding the inactive β-lactamase may have at least 90%sequence identity to a sequence of any one of SEQ ID NOS: 2-7, whereinnucleotides 202 to 204 of the sequence of any one of SEQ ID NOS: 2-7does not encode serine. This may be interpreted as, i.e., thepolynucleotide encoding an inactive β-lactamase may be translated to thesame protein sequence as that encoded by any one of SEQ ID NOS: 2-7, butthe polynucleotide may contain silent nucleotide mutations compared tothe sequence of any one of SEQ ID NOS: 2-7, where the silent nucleotidemutations do not change the amino acids encoded by the polynucleotide.

As used herein, the term “mutagenesis” refers a process in which thegenetic make-up of an organism (e.g., bacteria) is changed. In someembodiments, mutagenesis may be induced by certain environmentalconditions, i.e., growing E. coli cells at a restrictive temperatureand/or adding a mutagen in the cell culture.

As used herein, the term “solid media” refers a cell culture media thatis solid or semi-sold (e.g., a gel) and contains nutritional elementsbacteria need for growth. In some embodiments, the solid media may alsocontain certain selective markers, e.g., an antibiotic. In someembodiments, a solid media may be made from Luria-Bertani broth (LBbroth).

As used herein, the term “restrictive temperature” refers to atemperature that leads to an increase mutagenic frequency and acts as aselective pressure for the E. coli cells. In some embodiments,restrictive temperature may be about 37° C. In some embodiments, areversion event is more likely to occur under restrictive temperature.For example, an inactive β-lactamase having a S68P mutation may undergoa reversion event back to an active β-lactamase having serine atposition 68 instead of proline.

As used herein, the term “permissive temperature” refers to atemperature allows or encourages the normal growth of E. coli cells,without causing increase mutagenesis in the E. coli cells. In someembodiments, restrictive temperature may be about 30° C.

As used herein, the term “fluorescent protein” refers to a protein thatexhibits low, medium, or intense fluorescence upon irradiation withlight of the appropriate excitation wavelength. The fluorescentcharacteristic of fluorescent protein is one that arises from thechromophore, wherein the chromophore results from autocatalyticcyclization of two or more amino acid residues of the protein.

As used herein, the term “non-fluorescent protein” refers to a proteinthat can become fluorescent upon a reversion event. A non-fluorescentprotein may be constructed by mutating one or more nucleotides or one ormore amino acids of a fluorescent protein. Once the mutated nucleotideor amino acid is returned to the non-mutated version (the nucleotide oramino acid present in the initial fluorescent protein) after a reversionevent, the non-fluorescent protein can become fluorescent again.

As used herein, the term “mutagen” refers to a chemical agent thatcauses changes in the genetic make-up of an organism (e.g., bacteria)and increases the mutagenic frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1C: Reporter constructs. (A) TEMrev-GFP reporter. Mainfeatures: Lac promoter: 143-172; LacZ fusion: 217-288; Cycle 3 GFP:289-1005; kanamycin resistance: 1219-2004; β-lactamase: 2291-3151(2492-2494 S68); ColE1 plasmid origin of replication 3299-4091. In theTEMrev-GFPrev variant, the Q183R mutant codon is at positions 835-837.The mutant codon is CGA (R), which requires a G→A transition to revertback to CAA (Q). (B) sfGFPrev-TEM reporter. Main features: Ori 3999-479;sfGFP lac promoter 143-172; lacZ fusion 223-259, sfGFPrev: 201-1014; 12amino acid serine/glycine-rich linker 1015-1050; lactamase 1051-1911;M13 ori 1953-2462; kanamycin resistance (opposite orientation)3400-2575; and lactamase fragment 3553-3851. (C) Negative control notbearing the TEM1 gene.

FIGS. 2A and 2B: Methods for detection of two mutations separated intime. Colonies representing reversions in TEM1 β-lactamase were expandedunder restrictive (mutagenic) conditions, their plasmid pools wererecovered through miniprep and retransformed. The two reversionreporters are shown as star (S68X) and circle (Q183R). (A) Detection offirst mutation: once reversion at the S68X site occurs under selectivepressure, the reversion events get amplified, representing a majority ofthe plasmid population and leading to carbenicillin resistance. (B)Detection of second mutation: single, carbenicillin-resistant colonies(white arrow) are grown. Plasmids from these cultures are recovered.Reversions at the Q183 site of the GFP reporter are detected byretransformation of recovered plasmids into a readout strain, producingfluorescent colonies (white arrow) on a background on non-fluorescentones. The frequency of reversion can be used to estimate rate ofmutagenesis.

FIGS. 3A and 3B: sfGFPrev-TEM reporter containing the mutation K126stopin sfGFPrev on solid plates. Following Pol I mutagenesis, cells wereplated on LB carbenicillin. (A) Plasmids recovered from cells expressingLF-Pol I. (B) Plasmids recovered from cells expressing WT Pol I(control).

FIGS. 4A and 4B: LF-Pol I reversion profile. The set of six S68Xreporters was transformed in JS200 cells expressing LF-Pol I anderror-prone reporter plasmid replication was performed as described inmethods. As a control, the same reporters were transformed into JS200cells expressing WT Pol 1. (A) Original reversion frequencies in logscale. Error bars represent standard deviation of triplicates. (B)Reversion frequency relative to control cells expressing WT polymerase I(fold, in log scale)

FIGS. 5A-5D: Mutagenesis assay in 96-well format. Cells bearing twosample reporters, S68P (which detects C:G→T:A mutations) or S68R1 (whichdetects A:T→C:G and A:T→T:A mutations) underwent error-prone plasmidreplication as described in the examples. Cells were recovered bywashing the plates and inoculated into 96-deep-well plates to a final ODof 0.5. At different time points (shown in the X-axis) samples weredrawn and kept at 4° C. After completion of the time-course,fluorescence and optical density (OD600) were measured. LF-Pol Imutagenesis, triangles; WT Pol I control, squares, negative control withno β-lactamase gene, circles. (A) S68P reporter, OD600, in log scale.(B) S68R1 reporter, OD600, in log scale. (C) S68P reporter,fluorescence. (D) S68R1 reporter, fluorescence. Error bars representvariation between duplicates.

FIG. 6: Q183R reporter: R183Q reversion on a plate containing 9400colonies is shown. Transformation in DH5α cells (readout strain) ofplasmids recovered from expansion in liquid culture of a non-fluorescentcarbenicillin-resistant colony and grown under continuous mutagenesisconditions (diagrammed in FIGS. 2A and 2B). No revertants were seen in73,500 transformants from plasmids recovered in cells expressing WTpolymerase grown under the same conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure encompasses reporter constructs used for thedirect detection of mutagenesis in prokaryotes. In some embodiments, thereporter constructs use transcriptional coupling of β-lactamase (TEM-1)reversion and a fluorescent protein (e.g., GFP). The reporter constructsmay also employ translational fusion between β-lactamase (TEM-1) and afluorescent protein (e.g., GFP) containing a stop codon. In otherembodiments, the reporter constructs may be designed to monitorcontinuous mutagenesis and measure the mutagenesis rate in a mutatorstrain. The reporters described herein produce a quantitative output,and therefore can reduce the amount of test compounds and labor involvedin the performance of mutagenesis assays.

I. Mutagenesis Assays

Mutagenesis may be detected directly or indirectly. Direct detection ofmutagenesis can be performed in prokaryotes. Genotoxicity can also bedetected indirectly, through transcriptional fusion of a reporter geneto a promoter that is indicative of DNA damage, such as genes belongingto the SOS response (umuDC, sulA, recN, recA), alkA, or nrdA.Genotoxicity can also be detected physically by detecting DNA damage(breaks or rearrangements) using, e.g., Comet assay. In addition toprokaryotic systems, a variety of eukaryotic organisms, notably yeast,Drosophila, and mouse, have been used.

Relative to indirect methods of mutagenesis detection in prokaryotes,mutagenesis assays have the advantage of being able to detect specificchanges in DNA sequence rather than all DNA damage-induced alterationsin gene expression. Compared to eukaryotic model systems, bacterialassays are fast and cheap, but cannot detect mutagenesis in targets notconserved between prokaryotes and eukaryotes (such as cytoskeleton,nucleotide excision repair targets). Bacterial assays also cannot detectbioactivation as accurately. Still, direct mutagenesis assays inbacteria constitute one of three assays required by regulatory agenciesfor the demonstration of safety for potential clinical compounds. Theother two assays required are related to a eukaryotic cell culture testand an animal test.

General approaches for detecting mutagenesis in prokaryotes includereporter inactivation (measured in a forward mutation assay) andreversion of an inactivating mutation (measured in a reversion mutationassay). Both the forward mutation assay and the reversion mutation assayare labor-intensive, involving visual screening, quantification ofcolonies on solid media, and/or obtaining a Poisson distribution inliquid culture. Forward mutation assays are based on the inactivation ofa reporter. Reporters can produce colorimetric (e.g., galK, lacZ,luciferase), luminescent, fluorescent (e.g., GFP), or electrochemicalsignals. Inactivation can result from a variety of mutations. Thus,compared to reversion assays, forward mutation assays detect events thatare more frequent. Forward mutation assays also provide a more accuraterepresentation of the range of genetic changes induced by the relevantmutagen because inactivation is generally not dependent on a specificmutation occurring. In some cases, the readout for these involves asurvival marker, e.g., a gene that confers resistance to a drug or tothe presence or absence of a nutrient. RpoB (a gene encoding for RNApolymerase) is an example, as mutations in a variety of loci produceresistance to rifampin. AraD is another example. The cells used in thisassay have a mutation in the araD gene, which results in theaccumulation of a toxic intermediate when arabinose is present in thegrowth media. Mutations upstream of the araD gene that inactivate theoperon prevent the metabolism of arabinose, causing the accumulation ofthe toxic intermediate and making cells resistant to arabinose.Sectoring, which detects inactivation of a reporter within a colony asan indication of high and continuous mutation rates, is another variantof a forward mutation assay. Forward mutation assays are often morelabor-intensive because they require screening.

Reversion assays detect when a known inactivating mutation at apre-determined site is reverted to wild type, typically through aselection (auxotrophy, antibiotic resistance, FACS sorting). Theavailability of selection increases the sensitivity of these assaysrelative to the forward mutation assays described above and thereversion assays can also accurately detect a specific mutation.However, the dependence of reversion assays on individual mutations atpre-determined sites makes them susceptible to sequence context effectsand limits the range of genetic changes that can be detected. Thereadout for such an assay can be any of the signals described above(e.g., colorimetric, luminescent, fluorescent, or electrochemicalsignals). The Ames Test was the first reversion assay to be developedand it is still by far the most widely-used method for testingmutagenesis in prokaryotes. The Ames Test detects the reversion of amutation that prevents the biosynthesis of histidine and allows thegrowth of bacteria on solid agar in the presence of trace amounts ofhistidine. A set of six strains has been developed to detect a broadrange of point mutations and frameshift mutations. Two furthervariations have been developed to facilitate high-throughput formattingand to reduce the amount of sample needed. The Mini-Ames Test followsthe standard Ames Test protocol, except at a smaller size. The AmesFluctuation Test is performed in liquid culture, with the mutationdetected by a chromophore that indicates growth. Reversion assays basedon TEM β-lactamase have also been developed and one of the assaysincludes a set of six point mutations reporting on each type of pointmutation that is possible in double-stranded DNA. All these reversionassays produce a binary output, i.e., growth vs. no growth. As a result,determining the mutagenicity or genotoxicity of a single concentrationof a test compound requires fine-tuning the dose and serial dilutions toobtain countable colonies on solid plates or a sufficient number ofpositive wells that follow a Poisson distribution in a liquid culture.

Another type of reversion assay is the papillation assay, which can beused to detect alterations in mutagenesis rates in vivo. This assay isbased on a mutation in the gal2K gene, which renders cells unable toferment galactose. Cells are grown on Mac-Conkey-galactose plates,producing white colonies. Spottings on the surface of these colonies,often referred to as colored papilla (sectors), represent microcoloniesderived from a single Gal⁺ mutant capable of galactose fermentation. Theoutput is only semi-quantitative as it depends on mutation eventsoccurring early enough to allow for visual detection.

Mutator strains are bacterial strains that consistently exhibit anelevated mutation frequency and can be identified by their ability toproduce sectored colonies. There are some indications that the mutationrates in mutator strains are not constant, as there is acounter-selection against high mutation rates due to the deleteriousand/or adaptive effects of mutations. In addition, studying the dynamicsof mutagenesis in mutator strains using reporters is difficult becausemutations can inactivate the reporter regardless of its forward orreversion status with a probability that grows exponentially with thenumber of mutations present.

II. Revision Reporters

Disclosed are reporter systems that can be used to detect and quantifypoint mutations. These reporters are provided on a plasmid comprising apMB1 (ColE1-like) plasmid origin of replication. This has severaladvantages over a chromosomal location: (1) a plasmid reporter increasesthe number of targets for mutagenesis by at least one order ofmagnitude, since ColE1 plasmids are multicopy plasmids; (2) the factthat plasmids are present in multiple copies also allows amplificationof reporter signal through selection; and (3) a plasmid reporterfacilitates exposure to mutagens ex vivo, in this scenario,transformation would be performed only to obtain a readout.

TEM-1

TEM-1 is a type of β-lactamase found in Gram-negative bacteria.Expression of TEM-1 confers resistance to carbenicillin, as well asother β-lactam antibiotics such as penicillin. The version of TEM-1 usedthe reporters as disclosed herein can be inactivated through mutationsin the S68 position of the protein. S68 is a serine residue thatpolarizes the carbonyl group of the β-lactam amide bond in the β-lactamring of β-lactamase antibiotics and is completely intolerant to aminoacid changes. A set of six TEM-1 constructs with nucleotide pointmutations at S68 was engineered such that each nucleotide point mutationis within the serine-coding codon (nucleotides 202 to 204 (“AGC”) of SEQID NO: 1 encodes for serine). As a result, each of the 6 pairs ofnucleotide substitutions that are possible in duplex DNA can bedetected. Point mutations at this position were engineered to be onenucleotide away from a serine-coding codon so that each of the 6 pairsof nucleotide substitutions that are possible in duplex DNA can bedetected. Table 1 below lists the nucleotide sequence of wild-type TEM-1(SEQ ID NO: 1), the nucleotide sequences encoding the six TEM-1constructs with mutations at S68 codon (SEQ ID NOS: 2-7), and theprotein sequence of wild-type TEM-1 (SEQ ID NO: 8).

TABLE 1(bold nucleotides in SEQ ID NOS: 1-7 correspond to the codon codingfor the amino acid at position 68)SEQ ID NO: 1 (nucleotide sequence encoding wild-type TEM-1)   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 2 (nucleotide sequence encoding TEM-1(S68P))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GCCAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 3 (nucleotide sequence encoding TEM-1(S68T))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GACAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 4 (nucleotide sequence encoding TEM-1(S68R1))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GAGAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 5 (nucleotide sequence encoding TEM-1(S68stop))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GTGAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 6 (nucleotide sequence encoding TEM-1(S68N))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GAACACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 7 (nucleotide sequence encoding TEM-1(S68R2))   1ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT  61GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181GAAGAACGTT TTCCAATGAT GCGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841TCACTGATTA AGCATTGGTA ASEQ ID NO: 8 (protein sequence of wild-type TEM-1)   1MSIQHFRVAL IPFFAAFCLP VFAHPETLVK VKDAEDQLGA RVGYIELDLN SGKILESFRP  61EERFPMMSTF KVLLCGAVLS RIDAGQEQLG RRIHYSQNDL VEYSPVTEKH LTDGMTVREL 121CSAAITMSDN TAANLLLTTI GGPKELTAFL HNMGDHVTRL DRWEPELNEA IPNDERDTTM 181PVAMATTLRK LLTGELLTLA SRQQLIDWME ADKVAGPLLR SALPAGWFIA DKSGAGERGS 241RGIIAALGPD GKPSRIVVIY TTGSQATMDE RNRQIAEIGA SLIKHW

The six TEM-1 constructs with mutations at S68 codon (SEQ ID NOS: 2-7)detect the following mutations: SEQ ID NO: 2 (TEM-1 (S68P)) detectsC:G→T:A mutations; SEQ ID NO: 3 (TEM-1 (S68T)) detects A:T→T:Amutations; SEQ ID NO: 4 (TEM-1 (S68R1)) detects A:T→C:G and A:T→T:Amutations; SEQ ID NO: 5 (TEM-1 (S68stop)) detects G:C→C:G mutations; SEQID NO: 6 (TEM-1 (S68N)) detects A:T→G:C mutations; and SEQ ID NO: 7(TEM-1 (S68R2)) detects C:G→A:T mutations.

The sequences of the oligonucleotide primers used to introduce thenucleotide mutations at the S68 position of TEM-1 are shown in Table 2.

TABLE 2 (bold nucleotides correspond to the codoncoding for the amino acid at position 68) Mutation Forward Primer T_(M)Reverse Primer T_(M) S68P TTTCCAATGATGCCAACTTTTAAAGTT 54.6° C.ACTCACGTTAAGGGATTTTGGTCATGA 58.3° C. (SEQ ID NO: 13) (SEQ ID NO: 14)S68T TTCCAATGATGACAACTTTTAAAGT 51.6° C. ACTCACGTTAAGGGATTTTGGTCATGA58.3° C. (SEQ ID NO: 15) (SEQ ID NO: 16) S68R1 CCAATGATGAGAACTTTTAAA46.3° C. ACTCACGTTAAGGGATTTTGGTCATGA 58.3° C. (SEQ ID NO: 17)(SEQ ID NO: 18) S68stop TTCCAATGATGTGAACTTTTAAAGT 51.6° C.ACTCACGTTAAGGGATTTTGGTCATGA 58.3° C. (SEQ ID NO: 19) (SEQ ID NO: 20)S68N CCAATGATGAACACTTTTAAA 46.8° C. ACTCACGTTAAGGGATTTTGGTCATGA 58.3° C.(SEQ ID NO: 21) (SEQ ID NO: 22) S68R2 CCAATGATGCGCACTTTTAAA 52.2° C.ACTCACGTTAAGGGATTTTGGTCATGA 58.3° C. (SEQ ID NO: 23) (SEQ ID NO: 24)Q183R GCAGACCATTATCGACAAAATACTCCA 57.0° C. CGGAAATGTTGAATACTCATACTCTTCCT56.5° C. (SEQ ID NO: 25) (SEQ ID NO: 26)

TEMrev-GFP

The TEMrev-GFP reversion reporter comprises a TEM-1 gene (e.g., SEQ IDNO: 1) or a mutant thereof (e.g., any one of SEQ ID NOS: 2-7) positioned5′ to a GFP or a GFP derivative in an expression plasmid. In someembodiments, a linker may be placed between the TEM-1 and the GFP.Examples of linkers are described in detail further herein. One exampleof a GFP derivative is Cycle 3 GFP, a variant of GFP optimized forfluorescence in E. coll. Other fluorescent proteins that may be used inthe reporters and methods of the disclosure are described in detailfurther herein. The TEM-1 gene (e.g., SEQ ID NO: 1) or the mutantthereof (e.g., any one of SEQ ID NOS: 2-7) may be placed in the sameplasmid and co-transcribed with the GFP or GFP derivative (FIG. 1A)under the control of the same promoter or two different promoters. Usingthis system, once an inactivated TEM-1 (e.g., an inactivated TEM-1encoded by any one of SEQ ID NOS: 2-7) is reverted back to the activeTEM-1 (e.g., wild-type TEM-1 encoded by SEQ ID NO: 1), growth in thepresence of carbenicillin can be quantitatively detected usingfluorescence, which has a much wider dynamic range than turbidity,without the need for lysing the cells. As a result, this constructallows the monitoring of growth over time.

sfGFPrev-TEM

The sfGFPrev-TEM reporter comprises an inactivated GFP derivative(superfold GFP (sfGFP)) and a TEM-1 (e.g., SEQ ID NO: 1). Theinactivated GFP derivative (sfGFPrev) comprises a stop codon in the GFP,rending it non-fluorescent. In some embodiments, a linker may be placedbetween the sfGFPrev and TEM-1 (e.g., a 12-amino acid serine andglycine-rich linker) (FIG. 1B). SEQ ID NO: 12 below shows the sequenceof sfGFP-TEM fusion protein, in which sfGFP does not contain a stopcodon. Q at position 69, K at position 113, and K at position 126 of SEQID NO: 12 may be mutated to a stop codon to create sfGFPrev-TEMreporter, in which sfGFPrev contains a stop codon.

(squiggly: sfGFP (SEQ ID NO: 10); bold: linker; underlined: TEM-1)SEQ ID NO: 12

LVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTT GSQATMDERNRQIAEIGASLIKHW

Superfolder GFP (sfGFP) is a derivative of Cycle 3 GFP that includes twoadditional mutations selected for robustness to translational fusions.sfGFPrev is preferably located at the N-terminus of TEM-1 and ispreferably expressed in Top10 cells rather than JS200, AB1157, orGW7101. The introduction of a stop codon truncating GFP expressioninactivates both GFP and TEM-1, since TEM-1 is placed downstream ofsfGFP, resulting in a non-fluorescent, carbenicillin-sensitivephenotype. A TGA stop codon may be introduced into sfGFP at threedifferent positions: Q69, K113, and K126 (see SEQ ID NO: 10). Reversionof the stop codon in the mutated sfGFP back to the codon encoding theoriginal amino acid at the particular position (e.g., Q at position 69,K at position 113, and K at position 126 of SEQ ID NO: 10) may result influorescence and successful translation of the entire sfGFPrev-TEMreporter. Thus, the translated and functional TEM-1 may be able toprovide carbenicillin resistance to the cells.

TEMrev-GFPrev

The TEMrev-GFPrev reporter monitors mutagenesis in mutator strainsquantitatively. TEMrev-GFPrev comprises a Cycle 3 GFP inactivated via aQ183R (CAA to CGA) mutation, which reverts to functional Cycle 3 GFP inresponse to a C:G→T:A mutation, and a TEM-1 mutant (e.g., any one of SEQID NOS: 2-7). Thus, this reporter couples two reversion assays: TEM-1reversion and GFP reversion. This double set of markers allows thedetection of sequential hits, separating beyond double mutation eventsin time and facilitating the detection of changes in mutation rates overtime. The TEM-1 and the GFP may be included in the same plasmid andtheir expression can be driven by the same or different promoters. Insome embodiments, a linker may be placed between the TEM-1 and the GFP.An example of a method of using the reporter is shown schematically inFIGS. 2A and 2B. Colonies containing the plasmid are plated oncarbenicillin plates to identify reversion events in TEM-1 (i.e., amutant TEM-1 that is inactive (any one of SEQ ID NOS: 2-7) beingreverted back to the wild-type and active TEM-1 (SEQ ID NO: 1)).Non-fluorescent colonies (i.e., the colonies containing wild-type andactive TEM-1 and inactivated GFP) are picked and grown in liquidculture. The plasmid DNA from these cultures is recovered andretransformed into a readout strain (e.g., Top10 or DH5a) to identifyreversion events in GFP (e.g., non-fluorescent and inactivated GFPcontaining R at position 183 being reverted back to the fluorescent andactive GFP containing Q at position 183) (FIG. 2B). Under theseconditions, fluorescent colonies are likely to be the result of areversion event that occurred after carbenicillin reversion, unless thereversion was already present in one of the copies of the plasmid poolwhen the first mutation occurred. This alternate explanation can beruled out if the frequency of reversion is lower than one divided by thecopy number of the reporter plasmid.

The TEMrev-GFPrev reporter presents an alternative to papillation assaysfor the characterization of mutator strains. The main advantage is thatthe output in this case is quantitative rather than semi-quantitative,allowing head-to-head comparisons between different mutators and/orgrowth conditions. Different inactivating GFP mutations can beintroduced, depending on the mutagenic profile of the mutator strain.The chromophore-containing cyclized hexapeptide (amino acids atpositions 64 to 69) is a good target for an inactivating mutation with anarrow tolerance to alternative amino acids. Other possible mutations inCycle 3 GFP are described further herein (see, e.g., Table 4).

III. Fluorescent and Non-Fluorescent Proteins

The three reporters described above (TEMrev-GFP, sfGFPrev-TEM, andTEMrev-GFPrev) utilize a GFP or a derivative thereof in order to obtaina fluorescence readout of the reversion event. GFP facilitatesquantification of growth in vivo, producing a much stronger signal thanturbidity. Note that in FIGS. 5C and 5D, the results of fluorescence areshown in a logarithmic scale and that the ratio of background-to-signalis at least two orders of magnitude higher. Further, for a giventime-point, the result of GFP fluorescence is quantitative. Thedisclosed reversion assays report that LF-Pol I produces more C:G→T:Amutations relative to A:T→C:G and A:T→T:A mutations. In FIGS. 5C and 5D,the fluorescent signal is much stronger at 30 hour time-point for theC:G→T:A reporter so a measurement at this time-point may be proportionalto mutation frequency determined by plating. Relative to otherquantitative reporters, GFP has several advantages that make it idealfor continuous measurement in culture: (1) it is highly stable, (2) noaddition of an external substrate is necessary, (3) no cell lysis isrequired, and (4) it is less susceptible to substrate interference. Twolevels of signal amplification are used in the disclosed methods. First,plasmids are present in multiple copies in each cell, resulting ingreater total GFP expression. Second, TEM-1 revertants (cells withmutations that revert a mutant and inactive TEM-1 (e.g., a mutant TEM-1encoded by the sequence of any one of SEQ ID NOS: 2-7) back to awild-type and active TEM-1 (e.g., a wild-type TEM-1 encoded by thesequence of SEQ ID NO: 1)) have a growth advantage over mutant cells(i.e., cells containing a mutant and inactive TEM-1), increasing thenumber of cells in liquid culture at a given time in a mutator relativeto a control.

The sequences of Cycle 3 GFP (SEQ ID NO: 9) and superfold GFP (sfGFP)(SEQ ID NO: 10) are shown in Table 3 below. The bold amino acids in SEQID NO: 9 represent the 12 amino acids each of which may be mutated toinactivate Cycle 3 GFP (Table 4 below further describes specific mutatednucleotides). The bold amino acids in SEQ ID NO: 10 (Q69, K113, andK126) are the amino acids each of which may be mutated to a stop codon(e.g., TGA) to create an inactive and truncated sfGFP.

TABLE 3 SEQ ID NO: 9 (protein sequence of Cycle 3 GFP)MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDELYKSEQ ID NO: 10 (protein sequence of sfGFP)MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGIT HGMDELYK

Other fluorescent proteins may also be used in the reporters describedherein. Examples of fluorescent proteins are well-known in the art, see,e.g., Gert-Jan Kremers et al., J Cell Sci. 124:157, 2011 and Stepanenkoet al., Curr Protein Pept Sci. 9:338, 2008. Examples of fluorescentproteins include, but are not limited to, green fluorescent protein(GFP), yellow fluorescent protein (YFP), enhanced blue fluorescentprotein (EBFP), azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2,ECFP, CyPet, mKeima-Red, TagCFP, AmCyan1, mTFP1, Midoriishi Cyan,TurboGFP, TagGFP, Emerald, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz,Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange,Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer,DsRed2, mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin(RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, PeridininChlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, andmRaspberry.

To create an inactive or non-fluorescent version of a fluorescentprotein, in some embodiments, a stop codon may be introduced within theprotein sequence. For example, the bold amino acids in SEQ ID NO: 10(Q69, K113, and K126) indicate the amino acids each of which may bemutated to a stop codon (e.g., TGA) to create an inactive and truncatedsfGFP. Table 4 further lists the potential mutants and the mutatednucleotide that may be introduced into Cycle 3 GFP to create anon-fluorescent Cycle 3 GFP. A sequence of a non-fluorescent Cycle 3 GFPis shown in SEQ ID NO: 11, wherein Q at position 183 of SEQ ID NO: 9 ismutated to an R. Further, as described above, one or more amino acidswithin the chromophore-containing cyclized hexapeptide of a fluorescentprotein (i.e., amino acids at positions 64 to 69) may be mutated toproduce a non-fluorescent protein. One of skill in the art would havethe ability and knowledge to identify amino acids within the chromophoreof a fluorescent protein, see, e.g., Stepanenko et al., Biotechniques 51(5): 313, 2011, Sarkisyan et al., Sci Reports 2:608, 2012, and Gross etal., Proc Natl Acad Sci USA. 97 (22): 11990-11995, 2000.

SEQ ID NO: 11 (Cycle 3 GFP (Q183R)):MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYRQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDELYK

TABLE 4 Amino acid position of Nucleotide Original Original Mutant SEQID NO: 9 substitution Codon amino acid amino acid 48 G to A T

C C Y 49 A to G

CT T A 56 C to T C

A P L 61 T to A G

C V D 62 A to G

CT T A 89 C to T

CC P S 91 G to A G

T G D 92 A to G T

T Y C 103 A to T G

C D V 143 A to G T

C Y C 183 A to C CA

Q H 213 A to G G

A E G

In some embodiments, a reporter may contain a mutant TEM-1 (e.g., thesequence of any one of SEQ ID NOS: 2-7) joined to the N-terminus of afluorescent protein (similar to the TEMrev-GFP reporter describedabove). A reporter may also contain a mutant and inactive fluorescentprotein joined to the N-terminus of a wild-type TEM-1 (e.g., thesequence of SEQ ID NO: 1) (similar to the sfGFPrev-TEM reporterdescribed above). In some embodiments, a reporter may contain a mutantTEM-1 (e.g., the sequence of any one of SEQ ID NOS: 2-7) joined to theN-terminus or C-terminus of a mutant and inactive fluorescent protein(similar to the TEMrev-GFPrev reporter described above). In any of thereporters described herein, a linker may be optionally placed betweenthe TEM-1 or a mutant thereof and the active or inactive fluorescentprotein. Examples of linkers are described in detail further herein.

IV. Linkers

In some embodiments, a linker may be used as a linkage or connectionbetween a TEM-1 or a mutant thereof and an active or inactivefluorescent protein. The linker may be a peptide including, e.g., 3-200amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80,3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9,3-8, 3-7, 3-6, 3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200,10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200,60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or180-200 amino acids). In some embodiments, a linker may be a peptideincluding 8-16 amino acids (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 15amino acids). Suitable linkers are known in the art, and include, forexample, peptide linkers containing flexible amino acid residues such asglycine and serine. In certain embodiments, a linker can contain motifs,e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 27),GGSG (SEQ ID NO: 28), or SGGG (SEQ ID NO: 29). In certain embodiments, alinker can contain 2 to 12 amino acids including motifs of GS, e.g., GS,GSGS (SEQ ID NO: 30), GSGSGS (SEQ ID NO: 31), GSGSGSGS (SEQ ID NO: 32),GSGSGSGSGS (SEQ ID NO: 33), or GSGSGSGSGSGS (SEQ ID NO: 34). In certainother embodiments, a linker can contain 3 to 12 amino acids includingmotifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 35), GGSGGSGGS (SEQ ID NO:36), and GGSGGSGGSGGS (SEQ ID NO: 37). In yet other embodiments, alinker can contain 4 to 20 amino acids including motifs of GGSG (SEQ IDNO: 28), e.g., GGSGGGSG (SEQ ID NO: 38), GGSGGGSGGGSG (SEQ ID NO: 39),GGSGGGSGGGSGGGSG (SEQ ID NO: 40), or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO:41). In other embodiments, a linker can contain motifs of GGGGS (SEQ IDNO: 27), e.g., GGGGSGGGGS (SEQ ID NO: 42) or GGGGSGGGGSGGGGS (SEQ ID NO:43).

In other embodiments, a linker can also contain amino acids other thanglycine and serine, e.g., GSAGSAAGSGEF (SEQ ID NO: 44), GENLYFQSGG (SEQID NO: 45), or SACYCELS (SEQ ID NO: 46). In particular embodiments, thelinker is GSAGSAAGSGEF (SEQ ID NO: 44).

EXAMPLES Example 1—Brief Description of Experimental Protocol MaterialsTransformation and Mutagenesis

Competent cells: Top10, JS200-pHSG_WTPolA, and JS200-pHSG_EP1 PoIA

ColE1 vectors: pGFPck (Cycle 3; fluorescent), pGFPck (Q183R;non-fluorescent), sfGFP, and pGFPuv_KanR

Other materials: 500 mL centrifuge bottles, Eppendorf centrifuge 5810 R(Eppendorf), 50 mL conical tubes (Fisher Scientific, Cat. #1443222), 15mL culture tubes (E&K Scientific, Cat. #EK-62262), LB broth (FisherScientific, Cat. #BP1426-2), LB agar (Fisher Scientific, Cat.#BP1425-2), 100 mm×15 mm disposable Petri dishes (Fisher Scientific,Cat.# FB0875713), kanamycin solution (30 mg/mL, store at −20° C.),kanamycin (30 μg/mL) LB agar and broth, 1.5 mL microfuge tubes (E&KScientific, Cat. #280150), TropiCooler, Model 260014 (BoekelScientific), MaxQ 4000 shaker/incubator (Barnstead International), andwater-jacketed incubator (Forma Scientific)

Washing Plates

Materials: kanamycin (30 μg/mL) LB broth, plate spinner, plate spreader,ethanol (200 proof), Bunsen burner, spectrophotometer cuvettes (FisherScientific, Cat. #14955127), 1.5 mL microfuge tubes (E&K Scientific,Cat. #280150), and BioMate 3 Spectrophotometer (Thermo Scientific)

Readout (Plates)

Materials: kanamycin (30 μg/mL) LB agar and broth, carbenicillin (100pg/mL) LB agar and broth, 1.5 mL microfuge tubes (E&K Scientific, Cat.#280150), plate spreader, plate spinner, ethanol (200 proof), Bunsenburner, water-jacketed incubator (Forma Scientific), and UV Light

Readout (Liquid Culture Assay)

Materials: kanamycin (30 μg/mL) LB broth, carbenicillin (100 μg/mL) LBbroth, 3 mm diameter glass beads (Sigma-Aldrich, Cat. #Z265926), AirPoretape sheets (Qiagen, Cat. #2017-10-RP), 96-well round-bottomed deep-wellplates (Fisher Scientific, Cat. #10011-944), 96-well flat-bottomedblack-walled plates (Fisher Scientific, Cat. #82050-744), microtiterplate lids (Fisher Scientific, Cat. #82050-829), MaxQ 4000shaker/incubator (Barnstead International), SpectraMax M2e Fluorometricand Spectrophotometric plate reader, dual monochromators, and Absorbance200-1000 nm and excitation 250-850 nm (Molecular Devices)

Plasmid Recovery

Materials 15 mL culture tubes (E&K Scientific, Cat. #EK-62262), 1.5 mLmicrofuge tubes (E&K Scientific, Cat. #280150), and Nucleospin Plasmid(NoLid) kit (Macherey-Nagel, Cat. #740499.250)

Sequencing Plasmids of Interest

NanoDrop ND-1000 Spectrophotometer for DNA quantification (ThermoScientific), 0.6 mL microfuge tubes (E&K Scientific, Cat. #280060-S),and MacVector version 12.7.5 for sequence analysis (MacVector Inc.)

Methods Transformation of ColE1 Plasmids by Heat-Shock Method

Prepare a 5 mL overnight culture in a 15 mL culture tube in LB media forthe cell line of interest. If necessary, include selective antibiotic inthe media for the desired cell line. Expand this culture into a sterile1 L Erlenmeyer flask containing 500 mL of LB media with selectiveantibiotic. Incubate this flask at 30° C. or 37° C. (depending on thecell line; incubate JS200 cell lines at 30° C., and all others at 37°C.) with shaking (225 rpm) until exponential phase is reached(OD₆₀₀=0.4-0.6). Chill the flask containing the cells on ice for 20minutes. For best results, cells should be kept chilled at all times.Transfer the liquid cultures to 500 mL plastic centrifuge bottles andcentrifuge at 4000 rpm for 20 minutes at 4° C. Pour off supernatant, andresuspend the cell pellet in 50 mL of chilled calcium chloride solution(100 mM CaCl₂, 10 mM HEPES, 15% Glycerol, pH 7). Transfer theresuspended cells into a 50 mL conical tube. Centrifuge the cells at4000 rpm for 20 minutes at 4° C. Pour off supernatant, and resuspend thecell pellet in 50 mL of chilled calcium chloride solution, andcentrifuge the cells at 4000 rpm for 20 minutes at 4° C. (repeat 3times).

After third wash with calcium chloride solution, pour off supernatant,and resuspend the cells in 5 mL of chilled calcium chloride solution.Keep cells on wet ice and use immediately, or aliquot into 1.5 mLmicrofuge tubes and place on dry ice for storage at −80° C. Pipette 40μL of cells into 1.5 mL microfuge tube per transformation. Pipette 100μg of plasmid DNA into the tube containing the competent cells and mixwell by pipetting up and down. Incubate on ice for 30 minutes.Heat-shock the cells at 42° C. for 90 seconds on Tropicooler block.Place cells back on ice for 5 minutes. Add 1 mL of LB Broth to themicrofuge tube containing the cells and DNA. Allow the cells to recoverfor 30 minutes to 1 hour at 30° C. or 37° C. (depending on the cellline; incubate JS200 cell lines at 30° C., and all others at 37° C.)with shaking (225 rpm). Plate transformed cells by spreading 100 μL withsterile plate spreader onto pre-warmed LB agar plates containing 30μg/mL kanamycin. Allow the cells to grow overnight at either 30° C. or37° C.

Inducing Mutagenesis

For ColE1 on plasmids which have been transformed into JS200-pHSG_EP1PoIA cell strains, incubate at 37° C. to induce mutagenesis. Use thesame plasmids transformed into JS200-pHSG_WTPolA as control.

Washing Plates

Observe plate for bacterial colony lawn formation, which consists of ahigh density of colonies. Place plate on a plate spinner. Add 1 mL LBbroth containing 30 μg/mL kanamycin directly to the plate surfacecontaining bacterial growth. Use sterile plate spreader to collectcolonies into LB Broth. Tilt plate slightly of collect broth containingharvested colonies into one area, and transfer as much as possible intoa 1.5 mL microfuge tube. Repeat colony harvesting steps again. Collectsecond wash into the same 1.5 mL microfuge tube. Dilute plate washes1:20 directly in spectrophotometer cuvettes (950 μL media+50 μL platewash), and mix by pipetting. Measure OD₆₀₀ of diluted plate wash usingthe BioMate 3 spectrophotometer, and multiply the measurement by 20 toobtain the actual OD₆₀₀ of the undiluted plate wash. Normalize all platewashes to OD₆₀₀=1 prior to readout experiments.

Readout (Plates)

Pre-warm LB agar plates containing 30 μg/mL kanamycin and LB agar platescontaining 100 μg/mL carbenicillin in incubator set at 30° C. or 37° C.(depending on the cell line; incubate JS200 cell lines at 30° C., andall others at 37° C.). Plate 100 μL of plate washes to pre-warmed plates(all washes plated to both LB agar plates containing 30 μg/mL kanamycinand LB agar plates containing 100 μg/mL carbenicillin) at appropriatedilutions to yield countable colonies. Incubate at 30° C. or 37° C.(depending on the cell line; incubate JS200 cell lines at 30° C., andall others at 37° C.) overnight. Determine the number of colonies oneach plate. Use counts to determine CFU/mL of OD normalized cultures oneach type of selective media.

Determine percent of TEM β-lactamase S68 revertants by the formula:

% Reversion=[(CFU/mL(carbenicillin))/(CFU/mL(kanamycin))]*100

Readout (Liquid Culture Assay)

Aseptically place one sterile 3 mm diameter glass bead into each well ofa 96-well deep-well round-bottomed plate using sterilized forceps.Transfer 950 μL of LB broth containing 30 pg/mL kanamycin to one welland 950 μL of LB broth containing 100 μg/mL carbenicillin to anotherwell for each construct to be tested at each time point. Inoculate wellswith 50 μL of 1:10 diluted plate washes (final inoculation OD₆₀₀=0.05).Cover with AirPore tape sheet. Remove 200 μL of TO time point to 96-wellblack-walled clear-bottomed flat-bottomed plates, cover with sterileplate lid, place at 4° C. Cover deep-welled plate with sterile plate lidand place in incubator at 30° C. or 37° C. (depending on the cell line;incubate JS200 cell lines at 30° C., and all others at 37° C.) withshaking (325 rpm). At appropriate time points, remove 200 μL of culturefrom pre-assigned well to 96-well black-walled plates. Between timepoints, the black-walled plates should be stored at 4° C., and thedeep-welled plates should be incubated at the appropriate temperaturewith shaking. At the last time point, remove culture and un-inoculatedblank wells to black-walled plates. Read OD₆₀₀ and fluorescence (ex. 395nm, em. 509 nm) from black-walled plates on SpectraMax M2e Fluorometricand Spectrophotometric plate reader. Plot OD₆₀₀ versus time andfluorescence versus time for constructs under both kanamycin selectionand carbenicillin selection to estimate relative rates of TEMβ-lactamase S68 reversion.

Plasmid Recovery

Pick reversion colonies from LB agar plates containing 100 μg/mLcarbenicillin generated previously, and inoculate into 3 mL of LB brothcontaining 100 μg/mL carbenicillin. Grow cultures overnight at 30° C. or37° C. (depending on the cell line; incubate JS200 cell lines at 30° C.,and all others at 37° C.) with shaking (225 rpm). Harvest cells bycentrifugation at 11,000×g for 1 minute, pour off supernatant, andisolate plasmid DNA (miniprep) based on manufacturer's instructions.

Sequencing Plasmids of Interest

Quantify plasmid DNA yield and purity using NanoDrop Spectrophotometer.Open NanoDrop software (ND-1000, version 3.8.1), and select nucleic acidquantification. Place 2 μL of purified water on cleaned pedestal andlower arm to initialize spectrophotometer. Place 2 μL of elution bufferon cleaned pedestal and lower arm to blank spectrophotometer. Place 2 μLof sample to be quantified on cleaned pedestal and lower arm to measureabsorption spectrum between 220 nm and 350 nm. Transfer 0.5 to 1 μg ofplasmid DNA to 0.6 μL microfuge tube with appropriate label. Transfer 10μL of 5 μM sequencing primer to 0.6 μL microfuge tube with appropriatelabel. Send plasmid DNA and sequencing primer to sequencing facility.Assemble and analyze sequences using the program MacVector version12.7.5.

Other Experimental Notes

Pre-warming plates prior to plating cells is essential for efficientmutagenesis. Typical recovery per 2 mL of LB is about 1.5 mL of platewash. Experimenter must estimate dilution needed to achieve a totalnumber of colonies on plate between 30-300. This may require some trialand error. The results indicate that a dilution factor of 10⁻⁷ iseffective for all constructs and controls on kanamycin plates andpositive controls (WT β-lactamase) on carbenicillin plates and nodilution for negative controls on carbenicillin plates. For reporterconstructs on carbenicillin plates, dilutions may vary depending on theexpected reversion frequency, but generally range between no dilutionand a dilution factor of 10⁻³. Be sure to take note of the dilutionfactor used for each construct plated, as this will be used to calculateCFU/mL.

Sterilize forceps by dipping in ethanol and holding over flame until redhot. It is recommended to have replicates at each time point. Inoculatedifferent time points and different constructs and controls intoseparate wells. Inoculate each culture into both kanamycin wells andcarbenicillin wells. Leave at least three wells on each plateun-inoculated, to be used as blanks duringspectrophotometry/fluorimetry. For cell lines growing at 30° C., culturegrowth will be slower. Plan time points accordingly. Take 200 μL fromfresh wells at each time point. Do not resample wells that have alreadybeen sampled at previous time points. Sample well by stabbing themicropipette tip through the AirPore sheet. Use caution to avoiddisturbing/cross-contaminating wells containing later time points orblanks.

Example 2—Experimental Validation

To validate the reporter system, an error-prone Pol I plasmidreplication was used. This system is based on expression of anerror-prone variant of DNA polymerase I (Pol I) in JS200, a polAl2(temperature-sensitive) strain of E. coli. Shift of this strain to 37°C. makes J200 cells dependent on the activity of the error-prone variantof Pol I (low fidelity Pol I or LF-Pol I) for survival. Specifically,LF-Pol I performs ColE1 plasmid replication and processes of Okazakifragments during lagging-strand replication in both the plasmid and inchromosomal DNA. This variant bears three mutations that decrease itsreplication fidelity: I709N in motif A (broadening its active site),A759R in motif B (favoring its closed conformation), and D424A(inactivating its proofreading domain).

Overnight culture under restrictive conditions (37° C.) leads to anincreased mutation frequency in ColE1 plasmids by over three orders ofmagnitude in vivo, about 1 nucleotide substitution per 1.5 kb. This istrue for most of the plasmid sequence, where Pol I appears to becompeting with Pol III. These loads are higher in areas replicatedexclusively by Pol I: the 150 nucleotides immediately downstream of theRNA/DNA switch (leading-strand synthesis by Pol I), about 500nucleotides upstream of the RNA/DNA switch (gap-filling oflagging-strand synthesis by Pol I), and about 20 nucleotide patchescorresponding to areas of Okazaki fragment processing by Pol I. It isworth noting that LF-Pol I is partially dominant in vivo, as expressionof this polymerase still produces ColE1 plasmid mutagenesis atpermissive temperature or in polA WT strains, albeit with about 4 foldlower frequency relative to JS200 at restrictive temperature.

In terms of mutation spectrum, the mutation frequency of LF Pol I on asingle strand in vivo was estimated. The vast majority of mutations(>95%) are point mutations and can be grouped in four groups: mostfrequent: C→T transitions (60%); frequent: A→G and A→T (20 and 10% ofthe total), respectively); rare: G→T, G→A, and G→T, and extremely rare:T→C, T→A, A→C, and C→G. The observation of the very low frequency of T→Ctransitions indicates that mismatch repair appears to be intact in thesecells. Given that the reporter detects mutations in double-stranded DNA,i.e., in pairs of complementary mutations, the following ranking basedon frequency is expected: C to T/G to A (most frequent)>A to G/T to C, Ato T/T to A>G to T/C to A>T to G/A to C>G to C/C to G.

Example 3—Reversion Detection Using Six TEMrev-GFP Reporters

Six TEMrev-GFP reporters (e.g., each of the sequence of any one of SEQID NOS: 2-7 joined to the sequence of GFP), a TEM-GFP positive control,and a negative control not bearing the TEM1 gene (FIG. 1C) weretransformed into JS200 E. coli cells expressing LF-Pol I. As anadditional control, these plasmids were also transformed into a JS200expressing WT Pol I. After recovery at 30° C., cells were plated onto LBagar plates pre-warmed to 37° C. containing kanamycin, thus switchingthe transformants to restrictive conditions. Mutagenesis occurred duringgrowth overnight at 37° C.

For the TEMrev-GFP reporter, growth of the transformants overnightproduced a high density of colonies (near-lawn). These colonies wereharvested from the plate into about 1.5 ml of LB broth. Absorbance at600 nm was determined to normalize the washes to OD₆₀₀=1. Thesedilutions were used to plate kanamycin plates (at further dilution of1:10⁷) and carbenicillin plates at different dilutions, depending onreversion frequencies (between neat and 1:10³ dilutions). This timeplates were incubated overnight at 30° C. to minimize additionalmutagenesis. Following incubation, the number of colonies on each platewere counted, and this number was used to calculate the reversion ratefor each reporter (FIGS. 4A and 4B). Interestingly, fluorescence was notuniform across all carbenicillin-resistant colonies, possibly due to thepresence of additional mutations affecting GFP expression and/orfunction.

Three of the reporters produced between 10 and 100-fold higherbackground mutation frequencies in the control strain expressing WT PolI relative to the other three (FIG. 4A). The three reporters withgreater spontaneous mutation frequency are: S68stop (G:C→C:G), S68N(A:T→G:C), and S68R2 (C:G→A:T). Given that Pol I appears to compete withPol III for ColE1 plasmid replication, the observed increase inspontaneous mutation frequency could be due to an increase in thefraction of plasmids that are replicated by Pol I as a result of WT PolI overexpression. Indeed, Pol I appears to be more mutagenic than PolIII in vivo (particularly for transitions), and its fidelity can bemodulated by Pols II and IV.

Two pairs exhibit lower reversion frequencies than expected: A:T→G:C andG:C→T:G. This could be the result of sequence-context dependent effects,which can be in part due to differential efficiency of mismatch repair.The observation that S68R1, which detects A:T→C:G and A:T→T:A mutations,produces fewer reversions than S68T, which detects A:T→T:A alonedirectly confirms the impact of local sequence context on mutationrates. Overall, then, profiling a mutation spectrum using the disclosedTEMrev-GFP reporter gives a general idea of which types of pointmutations are favored, particularly if there is as strong bias for aspecific type.

For the sfGFPrev-TEM reporter containing K126stop mutation in sfGFPrev,JS200 cells expressing LF-Pol I and transformed with the sfGFPrev-TEMreporter that were plated at 30° C. produced a semi-lawn ofcarbenicillin-resistant, fluorescent colonies. Sequencing of 10 thesecolonies showed point mutations at the stop codon in all cases,producing an L (three times), W (three times), Q (twice), and Y (twice).In eight of these cases, the WT signal was still detectable, suggestingthat the plasmid carrying the K126 point mutation had not replaced allthe copies of the original K126 stop reporter. Cells expressing WT-Pol Ihad practically no colonies (FIGS. 3A and 3B)

Example 4—Growth and Fluorescence Emission Kinetics

Following mutagenesis, plates were washed with LB media and normalizedto OD₆₀₀=1.0 as described above. These cultures were used to inoculate96-well plates in a 1:20 dilution. The plates were deep-wellround-bottom plates with glass beads (to facilitate oxygenation) and afinal volume of 1 mL was added to each well. The plates were thencovered with AirPore breathable sheets, in order to protect againstcross-contamination and evaporation effects, while still allowing formicrobe growth under aerobic conditions. Cells were grown at 30° C. withshaking at 325 rpm. At different time-points, 200 μL of each culture wastransferred to a set of black-walled flat-bottomed 96-well microtiterplates and kept at 4° C. At the end of the experiment, these plates wereread on a fluorescence-enabled spectrophotometric plate reader forabsorbance at 600 nm to determine growth and for fluorescence (withexcitement λ=395 nm, and emission λ=509 nm). Results were then used toplot growth kinetics curves for each construct under each antibioticselection. FIGS. 5A-5D shows the growth and fluorescence emissionkinetics for two of the reporters, S68P (which detects C:G→T:Amutations; the sequence of SEQ ID NO: 2 joined to the sequence of GFP)(FIGS. 5A and 5C) and for S68R1 (which detects (A:T→C:G and A:T→T:Amutations; the sequence of SEQ ID NO: 4 joined to the sequence of GFP)(FIGS. 5B and 5D)

Example 5—Analysis of Double Reversion Events

For continuous mutagenesis detection, colonies expressing LF-Pol I andbearing the TEMrev-GFPrev reporter were plated under restrictiveconditions as described above, but at a higher dilution factor in orderto obtain individual carbenicilin-resistant colonies. Threenon-fluorescent colonies were picked, and grown in liquid culture underrestrictive conditions. The DNA from these cultures was recovered andretransformed into DH5α cells to identify R183Q (fluorescent) revertants(FIGS. 2A and 2B). A total of 11,100 colonies from these threetransformations were obtained. Of these, 2 colonies in two separatetransformations exhibiting bright fluorescence (one of them is shown inFIG. 6) were found. Control plasmids expressing WT Pol I produced 73,500colonies, none of which exhibited fluorescence based on visualinspection.

A reversion frequency at the Q183R site (revert R183 back to Q183) wasfound to be about 1 in 10⁴ cells. This frequency is 2-3 fold lower thanthat observed at the TEMrev site (revert a mutant TEM-1 back to awild-type TEM-1). It is unclear which amino acid substitutions areallowed at this site, but a C:G→T:A mutation reverts R at position 183back to Q. Given that C:G→T:A mutations are the predominant mutationsintroduced by LF-Pol I, it can be assumed that this is the main mutationdriving the reversion.

To control for the possibility that R183Q reversions were alreadypresent in one of the copies of the plasmid pool of the original colony,the original carbenicillin-resistant colonies were expanded underpermissive conditions as well. Only 2 fluorescent colonies were observedin 9,300 transformants, and no fluorescent colonies were observed inplasmids recovered from cells expressing WT Pol I (113,000transformants). Given that the average plasmid copy number for thereporter plasmid in LF-Pol expressing cells is less than ten plasmidsper cell, these results confirm that the observed fluorescent coloniesare most likely the result of mutations at the 183 position of GFP thatoccurred after the P68S reversion.

A reversion frequency that is far lower than 1 divided by plasmid copynumber confirms that the revertants were not present in the cell wherethe original reversion of the TEM-1 marker occurred, and thereforeargues that the GFP reversion occurred at a later time-point. Thisobservation has two additional implications: (1) it suggests that underrestrictive conditions, LF-Pol I-expressing cells continue to generatemutations after a first passage, albeit at a reduced rate relative toearly culture; (2) it also suggests that at the permissive temperature,where the mutation rate is already low, mutation rates can be sustainedover longer periods of time. Random mutagenesis systems that maintainmutation rates over time would greatly facilitate directed evolution.Thus, the disclosed reporter can be used to fine-tune existing mutatorstrains such as XL-1 red, the MP6 mutagenesis system, or strains withaltered dNTP pools to identify conditions supporting constant mutationrates over time.

What is claimed is:
 1. A method of detecting mutagenesis in E. coli, themethod comprising: (a) culturing E. coli cells in a first liquid cultureat a restrictive temperature, wherein the E. coli cells in the firstliquid culture comprise a plasmid, wherein the plasmid comprises (i) afirst polynucleotide encoding a non-fluorescent protein, and (ii) asecond polynucleotide encoding an active β-lactamase, wherein the firstpolynucleotide and the second polynucleotide are operably linked to apromoter; (b) plating the E. coli cells in the first liquid culture on asolid media comprising an antibiotic; (c) incubating the first solidmedia at a permissive temperature that allow the growth of E. colicolonies; (d) selecting a fluorescent E. coli colony from the solidmedia; (e) culturing the fluorescent E. coli colony in a second liquidculture at a permissive temperature, wherein the second liquid culturecomprises the antibiotic; and (f) measuring the change in fluorescenceof the second liquid culture relative to the first liquid culture,wherein the change in fluorescence indicates mutagenesis of thenon-fluorescent protein to a fluorescent protein.
 2. A method ofdetecting mutagenesis in E. coli, the method comprising: (a) culturingE. coli cells in a first liquid culture at a restrictive temperature,wherein the E. coli cells in the first liquid culture comprise aplasmid, wherein the plasmid comprises (i) a first polynucleotideencoding an inactive β-lactamase and having at least 90% sequenceidentity to a sequence of any one of SEQ ID NOS: 2-7, whereinnucleotides 202 to 204 of the sequence of any one of SEQ ID NOS: 2-7does not encode serine, and (ii) a second polynucleotide encoding anon-fluorescent protein, wherein the first polynucleotide and the secondpolynucleotide are operably linked to a promoter; (b) plating the E.coli cells in the first liquid culture on a first solid media comprisingan antibiotic; (c) incubating the first solid media at a permissivetemperature that allow the growth of E. coli colonies; (d) selecting anon-fluorescent E. coli colony from the first solid media; (e) culturingthe non-fluorescent E. coli colony in a second liquid culture at arestrictive temperature, wherein the second liquid culture comprises theantibiotic; (f) plating the E. coli cells in the second liquid cultureon a second solid media comprising the antibiotic; (g) incubating thesecond solid media at a permissive temperature that allow the growth ofE. coli colonies; (h) selecting a fluorescent E. coli colony from thesecond solid media; (i) culturing the fluorescent E. coli colony in athird liquid culture at a permissive temperature, wherein the thirdliquid culture comprises the antibiotic; and (j) measuring the change influorescence of the third liquid culture relative to the first liquidculture, wherein the change in fluorescence indicates mutagenesis of theinactive β-lactamase to an active β-lactamase and of the non-fluorescentprotein to a fluorescent protein.
 3. The method of claim 1, wherein thefirst polynucleotide is located 5′ to the second polynucleotide in theplasmid.
 4. The method of claim 1, wherein the plasmid further comprisesa linker between the first polynucleotide and the second polynucleotide.5. The method of claim 1, wherein the antibiotic is kanamycin orcarbenicillin.
 6. The method of claim 1, wherein the β-lactamase isTEM-1.
 7. The method of claim 1, wherein the fluorescent proteincomprises GFP or a derivative thereof.
 8. The method of claim 1, whereinthe fluorescent protein comprises a sequence of any one of SEQ ID NOS: 9and
 10. 9. The method of claim 1, wherein the non-fluorescent proteincomprises a sequence of SEQ ID NO:
 11. 10. The method of claim 1,wherein the first polynucleotide and the second polynucleotide areexpressed as a fusion protein.
 11. The method of claim 1, wherein thefirst polynucleotide and the second polynucleotide are expressed as afusion protein comprising a sequence of SEQ ID NO:
 12. 12. The method ofclaim 1, further comprising exposing the E. coli cells to a testcompound added to the first liquid culture.
 13. The method of claim 2,further comprising exposing the E. coli cells to a test compound addedto the first and second liquid cultures.
 14. The method of claim 12,wherein the test compound is a mutagen.
 15. A kit comprising a plasmidcomprising (i) a first polynucleotide encoding an inactive β-lactamaseand having at least 90% sequence identity to a sequence of any one ofSEQ ID NOS: 2-7, wherein nucleotides 202 to 204 of the sequence of anyone of SEQ ID NOS: 2-7 does not encode serine, and (ii) a secondpolynucleotide encoding a fluorescent protein, wherein the firstpolynucleotide and the second polynucleotide are operably linked to apromoter.
 16. The kit of claim 15, wherein the E. coli cells are of amutator strain and/or a readout strain.
 17. The kit of claim 15, whereinthe first polynucleotide is located 5′ to the second polynucleotide inthe plasmid.
 18. The kit of claim 15, wherein the plasmid furthercomprises a linker between the first polynucleotide and the secondpolynucleotide.
 19. The kit of claim 15, wherein the β-lactamase isTEM-1.
 20. The kit of claim 15, wherein the fluorescent proteincomprises GFP or a derivative thereof.
 21. The kit of claim 20, whereinthe fluorescent protein comprises a sequence of any one of SEQ ID NOS: 9and 10.